Spotted fever rickettsioses are re-emerging throughout the world. These are tick-transmitted bacterial infections that are severely underreported. Infection of Rickettsia's main target, the vascular endothelium, triggers endothelial activation of a proinflammatory phenotype and various degrees of damage that result in leakage of intravascular fluid into all organs and activation of coagulation. All data of the response of the endothelium to rickettsial infection has been generated exclusively in vitro with endothelial cell lines and primary endothelial cells derived from large vessels cultured under static conditions. The problem is that endothelial cells cultivated in vitro are very different from endothelial cells in vivo since their phenotype is physiologically regulated by the tissue microenvironment and by constant blood flow on the luminal side. On the other hand, work on mice has been performed under the assumption that the mouse endothelium correctly models the human endothelium;however, this claim has not been substantiated. The lack of in vivo experimental systems of rickettsial infection that focus on human endothelial cells of the microvasculature is a major need that must be addressed to fill the gap in our understanding of the in vivo role of the endothelium in rickettsial pathogenesis. Thus, our objective is to produce a relevant in vivo experimental model of the human endothelium and characterize the phenotype in response to rickettsial infection. Our central hypothesis is that the transcriptional response of the mouse endothelium in response to rickettsial infection in vivo is unlike that of the human endothelium in vivo and very different from the response of the endothelium in vitro under static conditions. The two specific aims to test our hypothesis are: 1) Produce an experimental in vivo model of the human endothelium infected with Rickettsia;and 2) Identify the differences between in vitro and in vivo endothelial cells infected with Rickettsia. The contribution of our research will be twofold: 1) a detailed understanding of the in vivo phenotype of human endothelial cells in response to infection with Rickettsia;and 2) a critical assessment of the validity of in vitro studies of the endothelium and of the mouse endothelium as a model of the human endothelium responding to rickettsial infections. These contributions are significant because they will provide validated models to study the role of the endothelium in rickettsial pathogenesis and a detailed molecular knowledge of the response of the human endothelium to rickettsial infection. The innovation of our research lays in bridging the gap between animal and human experimentation by implementing novel in vivo physiological experimental systems of the human endothelium and our ability to focus on the transcriptome of the target cells of Rickettsia without the signal dilution produced by the more commonly used transcriptional profiling of whole tissues. While advancing the fields of endothelial biology and rickettsial immunology, our results will generate a relevant model for discovering and testing therapeutic strategies to control advanced rickettsial infections where antibiotics alone will not be sufficient. PUBLIC HEALTH RELEVANCE: In support of the mission of the NIH to improve health, we will refine our understanding of the response of the human endothelium to rickettsial infection. This will open the field for the discovery of novel targets of intervention that may be used to control advanced infections when antibiotics alone will not prevent major complications and fatalities. The knowledge generated here will not only contribute to the fields of endothelial biology and rickettsial immunology, but will also impact other fields where vascular endothelial cells play an important role in pathogenesis such as atherosclerosis, cancer, autoimmune vasculitides, and solid organ transplantation.